Protein-protein interactions (PPIs) play a key role in defining protein functions in biological systems. Aberrant PPIs can have drastic impacts on basic cellular processes, and thus lead to various human diseases. PPI interfaces have recently become a new paradigm for therapeutics. Therefore, mapping PPIs and their interaction interfaces in living cells is not only fundamental to understanding protein function and regulation, but also vital to identifying potential targets for better therapeutics. Previously we have developed an integrated approach termed QTAX to effectively capture and identify static and dynamic protein interactions in order to provide a more authentic snapshot of protein interaction networks as they exist in living cells. To advance the study of in vivo PPIs beyond interaction identities, we have successfully developed an in vivo cross-linking mass spectrometry (XL-MS) platform based on a novel membrane permeable, enrichable and MS-cleavable cross-linker, i.e. Azide-A-DSBSO, and multistage tandem mass spectrometry (MSn). This strategy has been successfully applied to map PPIs at the proteome scale and the protein complex level. This technological advancement signifies a leap forward in studying in vivo PPIs and offers an essential framework for us to further develop novel technologies towards the ultimate goal of defining in vivo dynamics of protein complexes at the systems level. The 26S proteasome is the protein complex responsible for ubiquitin/ATP dependent protein degradation. Dysregulation of proteasomal degradation has been implicated in many diseases. Up to now, the high-resolution structure of the 26S proteasome remains unresolved and its regulation is poorly defined. Therefore, detailed structural analysis of the human 26S proteasome complex will be critical to uncover molecular details underlying its function and regulation. To this end, we propose 1) to develop the next generation of multifunctional cross-linkers for defining PPIs of protein complexes in living cells, 2) to develop a quantitative XL-MS strategy for charting in vivo dynamics of the human 26S proteasome upon H2O2-induced oxidative stress, 3) to dissect the interaction dynamics of the human 26S proteasome and its associated deubiquitinases. The proposed experiments will not only result in an exciting technological advancement in proteomics research, but also help address important yet unresolved biological questions associated with proteasomal biology and its relevance to human diseases.